Toxicologic Pathology

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Comparison of Cardiac Troponin I and T, Including the Evaluation of an Ultrasensitive Assay, as Indicators
of Doxorubicin-induced Cardiotoxicity
William J. Reagan, Malcolm York, Brian Berridge, Eric Schultze, Dana Walker and Syril Pettit
Toxicol Pathol 2013 41: 1146 originally published online 26 March 2013
DOI: 10.1177/0192623313482056
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Biomarkers
Toxicologic Pathology, 41: 1146-1158, 2013
Copyright # 2013 by The Author(s)
ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1177/0192623313482056

Comparison of Cardiac Troponin I and T, Including

the Evaluation of an Ultrasensitive Assay, as
Indicators of Doxorubicin-induced Cardiotoxicity
WILLIAM J. REAGAN1, MALCOLM YORK2, BRIAN BERRIDGE3, ERIC SCHULTZE4, DANA WALKER5, AND SYRIL PETTIT6
1

Pfizer Global Research Development, Groton, Connecticut, USA

2
GlaxoSmithKline, Hertfordshire, United Kingdom
3
GlaxoSmithKline, Research Triangle Park, North Carolina, USA
4
Department of Pathology, Lilly Research Laboratories, Indianapolis , Indiana, USA
5
Global Pharmacovigilance and Epidemiology, Bristol-Myers Squibb, Wallingford, Connecticut, USA
6
Health and Environmental Sciences Institute, Washington, D.C., USA
ABSTRACT
Cardiac troponin (cTn) has been utilized to assess acute myocardial injury, but the cTn response in active/ongoing chronic injury is not well
documented. The purpose of this study was to characterize the cardiac troponin I (cTnI), cardiac troponin T (cTnT), high-sensitivity cTnI, hematology, and clinical chemistry responses in rats treated with doxorubicin. Rats treated with 1, 2, or 3 mg/kg/week (wk) of doxorubicin for 2, 4, or 6 wks
were sacrificed after 0, 2, or 4 wks of recovery and compared to untreated controls and animals treated with doxorubicin/dexrazoxane (50 mg/kg/wk)
or etoposide (1 and 3 mg/kg/wk). The incidence and mean magnitude of cTn response increased with increasing dose and/or duration of doxorubicin
treatment. Conversely, dexrazoxane/doxorubicin was partially protective for cardiotoxicity, and minimal cardiotoxicity occurred with etoposide
treatment. Both cTnI and cTnT effectively identified doxorubicin-induced injury as indicated by vacuolation of cardiomyocytes of the atria/ventricles. The association between the cTn responses and histological changes was greater at the higher total exposures, but the magnitude of cTn response
did not match closely with histologic grade. The high-sensitivity cTnI assay was also effective in identifying cardiac injury. Alterations occurred in
the hematology and clinical chemistry parameters and reflected both dose and duration of doxorubicin treatment.
Keywords:

cardiovascular system; clinical pathology; rat; cardiac troponin; doxorubicin.

INTRODUCTION

ongoing cardiac myodegeneration and necrosis (Berridge et al.

2009; OBrien 2006, 2008; Wallace et al. 2004). In support of
this, several published experimental studies in laboratory species
have characterized the response of cTn to chemically induced

In preclinical safety assessment, cardiac troponin (cTn), a

successful translational biomarker for human cardiotoxicity, is
becoming more commonly utilized as a biomarker of active/

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was conducted under the
auspices of the ILSI Health and Environmental Sciences Institute (HESI). HESI is a public, nonprofit foundation whose mission is to engage scientists from academia,
government, and industry to identify and resolve global health and environmental issues. HESI receives support primarily from its industry sponsors. The opinions
expressed herein are those of the authors and do not necessarily represent the views of HESI. Contributing Organizations from the HESI Cardiac Biomarker Working
Group and Application of Genomics to Mechanism-Based Risk Assessment Technical Committee: Abbott Laboratories, Actelion Pharmaceuticals Ltd., Allergan Inc.,
Amgen Inc., Astellas Pharma Inc., AstraZeneca Pharmaceuticals, Auburn University, Bayer HealthCare Pharmaceuticals, Berlex Labs, Biogen Idec MA Inc., Boehringer
Ingelheim, Bristol-Myers Squibb Company, Bundesinstitut fuer Arzneimittel und Medizinprodukte, Cornell University, Covance Laboratories Inc., Daiichi Sankyo Co.
Ltd., Data Sciences International, Dow Chemical Co., Eli Lilly and Company, European Medicines Agency, Genentech, Inc, Georgetown University, GlaxoSmithKline,
Health Canada, Hennepin County Medical Center, Hoffmann-La Roche, Inc., Institute de Recherches Internationales SERVIER, Johnson & Johnson, Lifespan Heart
Center, Maastricht University, Merck & Co. Inc., Michigan State University, Mitsubishi Tanabe Pharma, Meiji Seika Pharma Co., Ltd., Novartis Pharmaceuticals, Pfizer
Inc., Pharmaceuticals & Medical Devices Agency, Sanofi, Sumimoto, Syngenta Ltd., Taiho Pharmaceuticals, Takeda Pharmaceutical Company Limited, Unilever PLC,
University of California, University of Minnesota, University of Minnesota Duluth Medical School, U.S. Army, U.S. Department of Agriculture, U.S. Environmental
Protection Agency, U.S. Food and Drug Administration, U.S. National Institute of Environmental Health Sciences, Vertex Pharmaceuticals Incorporated.
Address correspondence to: William J. Reagan, Drug Safety Research and Development, Worldwide Research and Development, Pfizer Global Research
Development, Eastern Point Rd., MS 274/1203, Groton, CT 06340, USA; e-mail: william.j.reagan@pfizer.com.
Abbreviations: AL, autolysis; AT, atrial thrombosis; BNP, brain natriuretic peptide; cTn, cardiac troponin; cTnI, cardiac troponin I; cTnT, cardiac troponin T;
EDTA, ethylenediaminetetraacetic acid; FH, focal hemorrhage; FN, focal necrosis; HCT, hematocrit; HESI, Health and Environmental Science Institute; HGB,
hemoglobin; IP, intraperitoneal; IV, intravenous; LOD, limit of detection; MD, myocellular degeneration; MFN, multifocal necrosis; MI, minimal; NHP,
nonhuman primate; N, number; NR, no result; NS, no sample; RBC, red blood cell; SD, Sprague-Dawley; VE, valvular endocarditis; WNL, within normal limits.
1146
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CARDIAC TROPONIN AND DOXORUBICIN CARDIOTOXICITY

acute myocardial injury. Isoproterenol, a nonselective b-adrenergic agonist, is one of the more common acute toxicants utilized
(Bertinchant et al. 2000; Brady et al. 2010; OBrien 2008; Tonomura et al. 2009; York et al. 2007; Zhang et al. 2008); in studies
in rats, the kinetics of circulating cTn are proportional and
predictable with the magnitude of the peak cTn response being
dependent on the dose of isoproterenol and sampling time.
Typically, after a single isoproterenol dose, a response can
be detected within an hour post exposure in rats, peaking within
a few hours and subsiding within 24 to 48 hr post exposure.
Histologically, injury can be detected within hours following
exposure and is characterized first by multifocal myofiber
hypereosinophilia. However, the peak histological severity
(myodegeneration and necrosis) does not typically occur
until 24 hr post dose (Clements et al. 2010). The kinetics of
cTn response in rats to acute myocardial injury have also
been documented with other toxicants, including treatment
with alcohol (Patel et al. 2001), phosphodiesterase inhibitors
(Zhang et al. 2006), and organophosphates (Yavuz et al. 2008).
Although studies that assess the kinetics of chemically
induced acute myocardial injury have been published, there are
few reported studies that have characterized the cTn response
with chemically induced chronic progressive myocardial
injury. Much of the investigative work with chronic myocardial
toxicity has been in studying the anthracyclines, such as doxorubicin (Balazs et al. 1981; Bertinchant et al. 2003; Herman
et al. 1998, 1999, 2001). Doxorubicin is widely used in the
treatment of cancer, but its use is specifically limited due to
the cardiotoxicity it can induce (Seiter 2005). The molecular
mechanisms for this toxicity as well as therapeutic approaches
to prevent the cardiotoxicity have been recently reviewed
(Octavia et al. 2012). Herman et al. (2001) demonstrated
that cardiac troponin T (cTnT) could be used to detect
doxorubicin-induced injury in rats, and concurrent dexrazoxane
treatment led to decreased severity of the cardiotoxicity as
assessed both by histologic examination of heart and by monitoring serum cTnT concentrations. Another study made a limited
time course comparison of cTnT concentration to echocardiography and brain natriuretic peptide (BNP) in rats treated with
doxorubicin weekly for 8 wks (Koh, Nakamura, and Takahashi
2004); animals were monitored for 6 to 12 wks, and showed
increases in cTnT concentration prior to an increase in BNP concentration in association with decreases in the ejection fraction.
Most of these studies of acute and chronic cardiotoxicity in
animals have not made direct comparisons between the utilization of cardiac troponin I (cTnI) versus cTnT. A single
published study of chronic cardiotoxicity in doxorubicintreated rats made a direct comparison of cTnI and cTnT and
showed that cTnT concentration correlated most closely with
the myocardial morphological changes including perivascular
and interstitial fibrosis as well as myocyte vacuolization
(Bertinchant et al. 2003).
The sensitivity and precision of the cTn assays have been
improved since many of these noted studies were published,
and newer research (not yet Food and Drug Administration
[FDA] approved) assays are now being evaluated to detect

1147

ultralow concentrations of cTn in humans (Morrow and

Antman 2009; Wilson et al. 2009). These high-sensitivity
assays can detect cTn in the low picogram per milliliter concentration in contrast to FDA-approved assays that are typically 10- to 100-fold less sensitive. These as yet unapproved
and research (high sensitivity) assays enable the detection of
baseline concentrations of cTn in normal/healthy people and
animals, and in myocardial injury that is below detectable limits
using the common FDA-approved assays (Apple and Collinson
2012; Morrow and Antman 2009; Sabatine et al. 2009). Low
concentrations of cTn detected with these high-sensitivity assays
may reflect normal physiological release of cTn from the minimal turnover of cardiomyocytes that has recently been proposed
to occur in humans (Bergmann et al. 2009). Recently, these
higher sensitivity assays have also been evaluated in the preclinical species used for safety assessment. Schultze et al. showed
that currently available research high-sensitivity assays have
utility with samples from rodents, dogs, and nonhuman primates
(Schultze et al. 2008). Schultze et al. also showed that in the
longitudinal assessment of cTn in rats that received an oral
gavage of vehicle, there was an increase in cTnI over time
relative to baseline concentrations (Schultze et al. 2009).
The purpose of this current investigation was to characterize
and compare the cTnI and cTnT response in a rat model of chemically induced chronic cardiac injury using FDA-approved
assays, as well as a high-sensitivity research cTnI assay. In this
investigation, serum analyte concentrations were assessed over
time in rats treated with a chronic low dose of doxorubicin,
with or without dexrazoxane, a known cardioprotective agent
(Herman et al. 2001; Hortobagyi 1997). Additional evaluated
animals were treated with etoposide, an antineoplastic topoisomerase inhibitor that is thought to be less cardiotoxic. Serum
cTnI and cTnT concentrations, as well as an assessment of in-life
observations, hematology and clinical chemistry parameters, and
cardiac histology were evaluated up to 6 wks with or without
recovery interval. The overall design and implementation of
this chronic doxorubicin study in rats was part of a larger study
performed by the Health and Environmental Sciences Institute (HESI) Technical Committee on Application of Genomics
to Mechanism-Based Risk Assessment to evaluate potential
association between gene expression data and mechanisms of
cardiotoxicity. The results of this other research will be reported
separately, although some investigative work on microRNA
profiling was recently published (Vacchi-Suzzi et al. 2012).
MATERIALS

AND

METHODS

Institutional Compliance Statement

The studies detailed in this article were approved by the
sponsor (HESI) and are consistent with the Guide for the Care
and Use of Laboratory Animals, the Animal Welfare Act, and
the Office of Laboratory Animal Welfare. Studies were
contracted to Covance Laboratories Inc. (Vienna, VA). The
animal facilities at Covance Laboratories, Inc., are accredited
by the Association for Assessment and Accreditation of
Laboratory Animal Care International.

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1148

REAGAN ET AL.

TOXICOLOGIC PATHOLOGY

TABLE 1.Treatment group designations and dose levels.

Group

Treatment

Study 1. Treatment phase

1
Control (vehicle)
2
Doxorubicin
3
Doxorubicin
4
Doxorubicin
5
Etoposide
6
Etoposide
7
Dexrazoxane
8
Dexrazoxane doxorubicin
Recovery phase
9
Control (vehicle)
10
Doxorubicin
11
Doxorubicin
Study 2. Treatment phase
12
Control (vehicle)
13
Doxorubicin
14
Doxorubicin
Recovery phase
15
Doxorubicin
16
Doxorubicin
Study 3. Treatment phase
17
Control (vehicle)
18
Doxorubicin
19
Doxorubicin
20
Doxorubicin
21
Etoposide
22
Etoposide
23
Dexrazoxane
24
Dexrazoxane doxorubicin
a

Na

Doxorubicin
(mg/kg/wk)

Dexrazoxane
(mg/kg/wk)

Etoposide
(mg/kg/wk)

Dose duration
(wk)

Termination
(wk)

6
6
6
6
6
6
6
6

0
1
2
3
0
0
0
2

0
0
0
0
0
0
50
50

0
0
0
0
1
3
0
0

2
2
2
2
2
2
2
2

2
2
2
2
2
2
2
2

6
6
6

0
2
3

0
0
0

0
0
0

2
2
2

6
6
6

6
6
6

0
2
3

0
0
0

0
0
0

4
4
4

4
4
4

6
6

2
3

0
0

0
0

4
4

6
6

6
6
6
6
6
6
6
6

0
1
2
3
0
0
0
2

0
0
0
0
0
0
50
50

0
0
0
0
1
3
0
0

6
6
6
6
6
6
6
6

6
6
6
6
6
6
6
6

Number of male rats.

Animals
Male, Crl:DC(SD) Sprague-Dawley rats (n 144), approximately 300 to 350 g body weight, were purchased from
Charles River Laboratories (Raleigh, NC). Rats were housed
individually in stainless steel cages. Environmental conditions
were defined as 18 to 26 C room temperature, 30 to 70%
humidity, with a 12-hr light/dark cycle. Rats were allowed to
access food (Certified Rodent Diet #8728C, Harlan, Teklad)
and drinking water ad libitum. Animals were given an acclimation period of at least 1 wk prior to study initiation and were
approximately 10 wks old at initiation of dosing. Rats were
identified by an implanted microchip bearing a unique number.
Selection of Animals
Rats were assigned to control or treatment groups using a
simple computerized randomization procedure to minimize
between-group differences in body weight.
Experimental Design
Three studies of various treatment lengths that incorporated
doxorubicin (an anthracycline antibiotic that works by intercalating DNA, inhibits topoisomerase II, and forms oxygen free
radicals), etoposide (an antineoplastic alkaloid that inhibits

topoisomerase II), and/or dexrazoxane (a cardioprotective

agent that chelates iron and decreases oxygen free radicals)
were used to better understand the kinetics of cardiac cTn
release in rat models of chronic cardiotoxicity. Group designations and dose levels are listed in detail in Table 1.
Study 1
Groups of 6 rats each received control vehicle (saline;
Groups 1 and 9), 1 mg doxorubicin/kg (Group 2), 2 mg doxorubicin/kg (Groups 3 and 10), 3 mg doxorubicin/kg (Groups 4
and 11), or 1 or 3 mg etoposide/kg (Groups 5 and 6, respectively) by intravenous (IV) injection via the tail vein. Group 7
consisted of 6 rats given 50 mg dexrazoxane/kg by intraperitoneal (ip) injection and Group 8 was comprised of 6 rats given
50 mg dexrazoxane ip followed by 2 mg doxorubicin/kg IV.
Control vehicle, doxorubicin, and etoposide were administered
IV once weekly for 2 consecutive wks. Dexrazoxane was administered ip once weekly for 2 consecutive wks (Groups 7 and
8). At 30-min post dexrazoxane administration, Group 7 rats
received saline IV and Group 8 rats received 2 mg doxorubicin/kg IV. Rats in Groups 1 to 8 were humanely euthanized
after 2 wks of treatment without a recovery period. Rats in
Groups 9 to 11 were treated for 2 wks, given 4 wks to recover,
and then were humanely euthanized.

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CARDIAC TROPONIN AND DOXORUBICIN CARDIOTOXICITY

Study 2
Groups of 6 rats each were given control vehicle (saline)
(Group 12), 2 mg doxorubicin/kg (Groups 13 and 15), or 3
mg doxorubicin/kg (Groups 14 and 16) IV once weekly for 4
wks. Rats in Groups 12 to 14 were humanely euthanized after
4 wks of treatment without a recovery period. Rats in Groups
15 and 16 were treated for 4 wks, allowed 2 wks to recover, and
then were humanely euthanized.
Study 3
Groups of 6 rats each received control vehicle (saline)
(Group 17), 1 mg doxorubicin/kg (Group 18), 2 mg doxorubicin/kg (Groups 19), 3 mg doxorubicin/kg (Groups 20), or 1 or 3
mg etoposide/kg (Groups 21 and 22, respectively) by IV injection via the tail vein. Group 23 consisted of 6 rats given 50 mg
dexrazoxane/kg by ip injection and Group 24 was comprised of
6 rats given 50 mg dexrazoxane ip followed by 2 mg doxorubicin/kg IV. Control vehicle, doxorubicin, and etoposide were
administered IV once weekly for 6 consecutive wks. Dexrazoxane was administered ip once weekly for 6 consecutive wks
(Groups 23 and 24). At 30-min post dexrazoxane administration, Group 23 rats received saline IV and Group 24 rats
received 2 mg doxorubicin/kg IV. All rats in this study were
humanely euthanized after 6 wks of treatment without a
recovery period.

Drug Formulation and Administration

Doxorubicin
Adriamycin, Lot No. 86G23FY (CAS# 23214-92-8) was the
generous gift from Adria Laboratories Inc. (Columbus, OH)
and was stored at room temperature (1530 C).
Etoposide
ETOPOPHOS1 Lot No. 5E04155 (CAS# 117091-64-2)
was purchased from Bristol-Myers Squibb (Princeton, NJ) and
was stored refrigerated (28 C) and protected from light.
Dexrazoxane
Zinecard1 Lot No. ADR074B (CAS# 24584-09-6) was purchased from Pharmacia/Pfizer (Kalamazoo, MI) and stored at
room temperature (1530 C).
Sodium Chloride (Vehicle Control)
For injection, 0.9% sodium chloride, United States Pharmacopeia, was purchased from Baxter Healthcare Corporation
(Deerfield, IL) and stored at room temperature (1530 C).
The doxorubicin and etoposide preparations were administered as an IV injection, in the tail vein of each rat each wk.
Individual doses were based on body weights and administered
in a 2-ml/kg dose volume. Dexrazoxane was administered as an
ip injection. Individual doses were based on body weights and
administered at a volume of 10 ml/kg.

1149

Mortality, In-life (Cageside) Observations, and Body

Weights
Morbidity, mortality, signs of injury, and access to food and
water were assessed twice daily for each animal on study. Body
weights were recorded upon arrival, on days 1, 8, 15, 29, 36, 43,
and just prior to necropsy.
Blood Collection
Approximately 0.5 ml of whole nonanticoagulated blood
was obtained via jugular puncture from all rats in Groups 1
to 4, 9 to 11, and 15 to 24 pretest and one time per wk for potential determination of the concentrations of cardiac troponins I
and T in sera. Prior to necropsy, rats were fasted overnight and
then anesthetized with 70% CO2/30% O2, and blood was
collected from the vena cava into tubes containing potassium
ethylenediaminetetraacetic acid (EDTA) for hematologic analysis or tubes with no anticoagulant for routine serum chemistry
and cTn determinations. EDTA blood samples for hematologic
analysis were chilled on wet ice and transported to the Clinical
Pathology Laboratory for immediate analysis. Whole blood for
serum biochemistry analysis and cTn determinations was
allowed to clot at room temperature for approximately 30 to
45 min before being spun in a centrifuge to obtain serum.
cTnI Concentration
Sera from control vehicle- and doxorubicin-treated rats were
analyzed for the concentration of cTnI using two methods: the
Access1 Immunoassay System (Beckman Coulter Inc., Brea,
CA) and/or the Verigene1 System High-sensitivity cTn I
Assay (Nanosphere Inc., Northbrook, IL). cTnT concentration
in serum was determined using Elecsys1 Troponin T Assay
(Roche Diagnostics, Nutley, NJ). For this study, the limit of
detection (LOD) for the two often-used cTn assays was 0.01
ng/ml (cTnTRoche) and 0.03 ng/ml (cTnIBeckman). A
value of 0.1 pg/ml was used for the newer investigative highsensitivity assay (cTnINanosphere).
Complete Blood Counts
EDTA blood samples were analyzed with the ADVIA 120
Hematology System (Siemens Healthcare Diagnostics, Deerfield, IL) using reagents from Bayer Diagnostics (Tarrytown,
NY) for the determination of erythrocyte count, hemoglobin
(HGB) concentration, hematocrit (HCT), mean corpuscular
volume, mean corpuscular HGB, mean corpuscular HGB
concentration, platelet count, total and differential leukocyte
counts, and reticulocyte count.
Clinical Chemistry Analytes
A Modular P Analyzer (Roche Diagnostics, Nutley, NJ) and
Roche reagents were used for determination of the concentrations of glucose, urea, creatinine, total protein, albumin,
globulin, cholesterol, triglycerides, total bilirubin, calcium,
inorganic phosphorus, sodium, potassium, chloride, and activities of alanine aminotransferase, alkaline phosphatase, sorbitol

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1150

REAGAN ET AL.

dehydrogenase, g-glutamyltransferase, aspartate aminotransferase, creatine kinase, and lactate dehydrogenase. The
albumin/globulin ratio was calculated. Isoenzymes of creatine
kinase were separated using the HYDRASYS Agarose Gel
Electrophoresis Apparatus and reagents (Sebia Electrophoresis, Norcross, GA).
Necropsy, Organ Weights, and Histopathologic
Examination
Rats were anesthetized by the inhalation of 70% CO2/30%
O2 and euthanized by exsanguination. Necropsy included a
macroscopic examination and organ (brain, heart, kidneys,
liver, and spleen) weights. Samples of heart, jejunum, kidneys,
liver (all lobes), gastrocnemius muscle, diaphragm, and the animal identification number were preserved in 10% neutral buffered formalin.
Each heart was longitudinally bisected from base to apex
along the middle of the ventricular free walls to reveal right and
left ventricles; right and left atria; both the atrioventricular
valves and the root of the aorta. One-half of the bisected heart
was placed in 4% formaldehyde/1% glutaraldehyde while the
other half was flash frozen for other studies. The fixed hemisections of heart were processed for methylmethacrylate plastic
embedment, sectioned at 1 to 2 mm, stained with toluidine blue
and examined histologically.
A representative histologic section of heart from each
animal was evaluated light microscopically for any evidence
of cardiomyocellular injury but more specifically for the cardiomyocellular vacuolation typical of doxorubicin-induced
cardiomyopathy (Bertinchant et al. 2003). Vacuolar changes
in ventricular and atrial cardiomyocytes were recorded
separately and semiquantitatively graded as minimal, mild,
moderate, or marked based on relative distribution of affected
cells. Qualitative grades were converted to ordinal scores to
facilitate recognition of trends in lesion severity. For example,
a minimal grade was given an ordinal score of 1. A mild
grade was given an ordinal score of 2, and so on. Ordinal
scores for the ventricles and atria were averaged for individual
animals and the mean scores averaged for the dose group
(Table 2). Lesions not typical for doxorubicin-induced cardiomyopathy were individually described capturing the character
of the lesion (e.g., necrosis vs. fibrosis), distribution, and
severity.
Statistical Evaluation
Statistical differences in hematology data, clinical chemistry data, body, and organ weights among experimental groups
were determined using one-way analysis of variance at the
5.0%, two-tailed probability level. Rank transformation was
conducted if Levenes test for variance homogeneity was
p  .05. Differences with p < .05 were considered significant.
Tabled results are expressed as mean values + standard deviation. Deming regression analysis was used to compare the cTnI
and cTnT results.

TOXICOLOGIC PATHOLOGY

RESULTS
Body Weight, Food Consumption, In-life Observations,
and Mortality
Decreases in mean body weight were observed in rats given
2 or 3 mg/kg/wk of doxorubicin (data not shown). The incidence and magnitude of the decreases relative to vehicle controls corresponded with those in mean food consumption and
were dose-dependent and duration-dependent. Body weight
decreases in animals given doxorubicin at 2 or 3 mg/kg/wk
were observed as early as wk 2 and were substantial by the end
of the 6-wk interval (mean weight decreases were 925% at 2
mg/kg and 1136% at 3 mg/kg below concurrent vehicle controls for wks 26). Body weight decreases in animals given 2 or
3 mg/kg showed only slight resolution by the end of the 4-wk
recovery interval. Considerably smaller differences in mean
body weight compared with concurrent vehicle controls were
seen in animals given 1 or 2 mg/kg/wk doxorubicin for only
2 wks, or dexrazoxane in combination with doxorubicin. No
notable body weight differences compared to vehicle controls
occurred with animals given dexrazoxane alone or etoposide.
A few deaths occurred at 3 mg/kg/wk after 4 wks (1 animal)
or 6 wks (4 animals) of doxorubicin administration in Groups
16 and 20, respectively. No notable treatment related in-life
findings were observed.
Hematology
Progressive alterations in the hematology data in animals
given doxorubicin, reflecting both dose and duration were
observed. These changes included decreased red blood cell
(RBC) count, HGB concentration, HCT and total leukocyte,
lymphocyte, and eosinophil counts relative to concurrent vehicle controls. Associated with these findings during the dosing
phase of the study was an increase in absolute reticulocyte and
platelet counts (Table 3). In the animals given doxorubicin at 3
mg/kg, changes were evident in all these parameters after 2 wks
dosing, while at the lower dose of 1 mg/kg at 2 wks only an
increase in reticulocyte counts was evident. Animals dosed at
2 mg/kg showed early changes at 2 wks (decreased total white
blood cell count, influenced mainly by a lowering of the lymphocyte count, and increased reticulocyte and platelet counts).
Near complete recovery toward concurrent vehicle control
levels was evident after 4 wks off dose in these animals at 2
mg/kg; but animals given 3 mg/kg/wk for 2 wks showed only
partial resolution in the reticulocyte, white blood cell, lymphocyte, and eosinophil counts, and progressive decreases of the
red cell mass parameters (RBC, HGB, and HCT) at the 4-wk
recovery interval. When animals were dosed for 4 wks at 2 and
3 mg/kg/wk followed by a 2-wk off dose period, there was no
evidence of recovery toward concurrent vehicle control levels
except in reticulocyte counts for animals given 2 mg/kg/wk.
In general, the alterations in rats given doxorubicin (2 mg/
kg) in combination with dexrazoxane (50 mg/kg) weekly were
similar to those animals receiving only doxorubicin but of a
lower magnitude of change after 6 wks dosing (Table 4). There

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1151

TABLE 2.Measurement of cardiac troponin (cTnT, cTnI) concentration in individual animals using the Nanosphere, Beckman Access, and Roche
analytical platforms following treatment with doxorubicin and correlation with cardiac histopathology.
cTn concentration
at week 6b

hs-cTnI
(Nanosphere) concentration
pg/mla
Dose
Control (Groups 9c and
17c combined)

1 mg/kg/week (Group
18c)

2 mg/kg/week (Groups
15d and 19c)

3 mg/kg/week (Groups
16d and 20c)

Animal
No.

Day 2

Week 4

Fold change
from day 2

487
488
489
490
491
492
535
536
537
538
539
540
M
541
542
543
544
545
546
M
523
524
525
526
527
528
547
548
549
550
551
552
M
529
531
532
533
534
553
554
555
556
557
558
M

1.6
3.5
3.8
2.6
1.7
1.7
1.0
0.8
3.5
0.7
1.0
2.6
2.04
1.0
2.9
1.6
2.1
1.9
1.1
1.77
1.0
2.3
2.6
4.8
2.0
2.9
1.0
2.4
1.2
1.9
1.2
6.2
2.46
1.0
3.6
1.0
3.4
3.5
2.3
2.7
2.3
1.8
2.8
0.4
2.25

3.9
3.5
2.3
1.6
3.5
1.9
1.9
1.1
1.5
1.0
0.3
0
1.88
1.0
0.1
0.1
0.1
2.4
5.1
1.47
9.9
5.6
9.0
0.3
6.3
9.8
3.9
4.1
4.0
11.4
16.7
20.4
8.45
4.2
66.4
12.7
12.1
17.7
11.1
14.2
67.0
8.6
17.8
7.3
21.74

2.4
0
0.6
0.6
2.1
1.1
1.9
1.4
0.4
1.5
0.3
0
1.11
1.0
0.03
0.05
0.05
1.3
4.7
1.18
9.9
2.4
3.5
0.06
3.2
3.4
3.9
1.7
3.3
6.0
14.0
3.3
4.54
4.2
18.5
12.7
3.6
5.1
4.8
5.3
29.1
4.8
6.4
18.3
10.24

Myocyte vacuolation
scores at week 6

cTnI
cTnT
(Beckman) ng/ml (Roche) ng/ml Atrial/ventricular means
0.02
0.07
0.220
0.050
0.030
0.030
0.150
0.050
0.190
0.040
NR
0.060
0.083
0.070
0.060
0.060
0.040
0.090
0.110
0.072
0.050
0.060
0.040
0.050
0.080
0.030
0.080
0.090
0.060
0.040
0.090
0.170
0.070
0.060
NS
0.060
0.050
0.030
0.180
NS
NS
NS
NS
0.670
0.175

0.010
0.019
0.073
0.010
0.010
0.010
0.049
0.011
0.073
0.010
0.013
0.010
0.025
0.038
0.020
0.028
0.010
0.030
0.050
0.029
0.039
0.050
0.026
0.017
0.058
0.010
0.109
0.082
0.042
0.037
0.074
0.190
0.061
0.090
NS
0.024
0.069
0.104
0.263
NS
NS
NS
NS
0.194
0.124

0.50
0.00
0.50
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.08
2.00
1.50
1.00
0.50
1.00
1.00
1.17
1.00
1.00
1.50
1.00
1.50
0.50
1.50
1.00
2.50
1.00
2.50
2.00
1.42
3.50
1.00
1.00
2.00
1.50
0.00
0.50
1.00
1.50
2.00
2.00
1.45

Comment

MI FN

FN

MI FN

FH and FN

FN
MI MFN
VE and AL
AT and MD

Note. AL autolysis; AT atrial thrombosis; cTn cardiac troponin; cTnI cardiac troponin I; cTnT cardiac troponin T; FH focal hemorrhage; FN focal necrosis; M mean;
MD myocellular degeneration; MFN multifocal necrosis; MI minimal; NR no result; NS no sample; VE valvular endocarditis; - within normal limits.
Lesion scores for atrial/ventricular vacuolation: minimal 1, mild 2, moderate 3, and marked 4.
a
Measurement of cTnI concentration was performed on in life samples collected on day 2 and week 4 obtained by jugular venipuncture.
b
Measurements of cTnI and cTnT concentrations were performed on samples collected from vena cava following terminal anesthesia.
c
Groups 9 (animals 487492), 17 (animals 535540), 18, 19 (animals 547552), and 20 (animals 553558) 6 weeks dosing.
d
Groups 15 (animals 523528) and 16 (animals 529534) 4 weeks dosing.

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1152

REAGAN ET AL.

TOXICOLOGIC PATHOLOGY

TABLE 3.Changes in selected hematology parameters (M and SD) following treatment with doxorubicin with or without recovery.a
Dose
mg/kg/wk

Weeks of dosing

2
6

2
6

2
2 4Rb
4
4 2Rb
6

2
2 4Rb
4
4 2Rb
6

Red blood cell

1012/L

Hemoglobin
g/dl

Hematocrit
%

Reticulocyte
109/L

Platelet
109/L

White blood cell

109/L

Lymphocyte
109/L

Eosinophil
109/L

8.8
0.204
8.51
0.255
8.86
0.355
7.74
0.504
8.17
0.56
7.98
0.816
7.08*
0.334
5.97
0.663
4.55
1.03
6.83*
0.509
6.34*
0.736
4.00*
1.112
5.51
0.342
2.64*
0.0771

16.5
0.5
15.5
0.42
16.6
0.68
14.6
1.1
15.5
1.03
14.8
1.63
13.3*
0.55
11.4
1.60
8.9*
2.17
12.9*
0.84
11.5*
1.83
7.3*
1.88
10.3
0.71
5.3*
2.19

48.1
3.08
44.6
1.53
48.4
3.6
41.9
3.45
45.3
4.25
43.3
5.06
37.7*
1.53
33.5
5.00
27.2*
6.55
36.6*
1.72
34.7*
4.65
20.6*
4.59
31.7*
2.43
16.8*
5.37

285.7
38.11
271.6
35.97
437.7*
200.1
421.5*
65.7
418.8*
66.67
253
23.41
654.4*
168.86
276.5
64.97
824.4*
174.73
561.6*
143.54
355.9
88.88
470.2
187.91
500.4
100.6
569.7*
150.33

1,247
198.3
1,260
119.3
1,217
402
1,784*
292.5
1,680
296.3
1,378
255.9
2,469*
321.6
1,942
515.1
2,457*
675.4
1,665
641.6
1,927*
422
2,790*
564.4
2,614
245.0
1,931*
397.4

9.15
1.763
10.68
1.337
7.35
2.065
7.23*
1.838
5.99
2.159
9.69
1.64
6.49*
1.459
7.87
3.206
5.21*
1.699
5.58
1.973
9.36
2.942
4.84*
0.693
4.23
1.246
4.08*
2.029

7.59
1.419
8.59
0.879
6.18
1.656
5.64*
1.347
4.76*
1.73
7.23
1.562
4.18*
0.795
4.38
1.795
2.67*
0.514
4.65
1.684
6.30
0.979
2.64*
0.785
2.27
1.049
1.57*
0.453

0.05
0.026
0.15
0.073
0.06*
0.023
0.06*
0.026
0.05*
0.05
0.15
0.031
0.04*
0.013
0.02
0.010
0.02*
0.013
0.01*
0.006
0.08
0.034
0.02*
0.089
0.01
0.004
0.00*
0.000

Note. M mean; SD standard deviation.

a
Results are expressed as mean values + SD (location below mean value). bRecovery phase. Number of animals per group 6, except n 5 for rats treated with 3 mg/kg/wk for 4
weeks with a 2 week recovery, and n 2 for rats treated with 3 mg/kg/wk for 6 weeks and no recovery.
*Significant at p  .05.

TABLE 4.Changes in selected hematology parameters (M and SD) following treatment with etoposide, dexrazoxane, and dexrazoxane in
combination with doxorubicin.a

Dose
mg/kg/wk
Etoposide 1

Weeks of
dosing
2
6

Etoposide 3

2
6

Dexrazoxane 50

2
6

Dexrazoxane 50 doxorubicin 2

2
6

Red blood
cell
1012/L
8.61
0.349
8.55
0.416
8.31*
0.275
8.53
0.336
8.71
0.260
8.88
0.602
8.11*
0.458
5.97*
0.737

Hemoglobin Hematocrit Reticulocyte Platelet

g/dl
%
109/L
109/L
16.4
0.82
15.1
0.59
16.0
0.72
15.5
0.51
16.5
0.56
15.8
0.93
15.6
0.82
12.2*
1.49

47.9
3.89
44.2
1.92
48.1
3.92
45.1
1.52
47.8
2.73
45.9
3.04
45.6
3.01
37.2*
4.38

348.8
45.22
322.2*
17.85
535.1*
47.80
422.0*
53.21
337.3
49.70
320.1*
21.44
600.1
36.13
864.4*
76.80

Note. M mean; SD standard deviation.

a
Results are expressed as mean values + SD (located below mean value). Number of animals per group 6.
*Significant at p  .05.

Downloaded from tpx.sagepub.com at University of Groningen on May 2, 2014

125.1
175.8
1,185
202.5
1,317
119.4
1,281
173.1
1,273
260.7
1,261
118.7
1,207
398.6
1,630*
246.1

White blood
cell
109/L
9.07
2.207
9.51
1.631
8.9
3.032
9.88
0.522
9.93
3.364
8.92
1.677
6.83
1.883
5.68*
0.924

Lymphocyte Eosinophil
109/L
109/L
7.67
2.112
7.19
1.313
7.39
2.570
6.95
0.569
7.85
2.685
6.85*
1.670
5.57
1.652
3.35*
0.791

0.11
0.024
0.14
0.098
0.06*
0.031
0.15
0.038
0.15
0.054
0.16
0.069
0.02*
0.005
0.02*
0.012

Vol. 41, No. 8, 2013

CARDIAC TROPONIN AND DOXORUBICIN CARDIOTOXICITY

1153

TABLE 5.Changes in selected clinical chemistry parameters (M and SD) following treatment with doxorubicin with or without recovery.a
Dose
Total protein Albumin Globulin Albumin: globulin Cholesterol Triglyceride Urea Creatinine Alkaline phosphatase
mg/kg/wk Weeks of dosing
g/dl
g/dl
g/dl
mg/dl
mg/dl
mg/dl
mg/dl
U/L
0

2
6

2
6

2
2 4Rb
4
4 2Rb
6

2
2 4Rb
4
4 2Rb
6

6.0
0.29
6.2
0.31
6.1
0.14
5.8
0.23
5.9
0.34
4.8
0.24
5.4*
0.20
5.4
0.29
5.1*
0.21
5.0*
0.32
4.6*
0.23
5.2*
0.23
5.4
0.34
5.1*
0.21

4.5
0.08
4.4
0.19
4.4
0.09
3.3*
0.75
4.0*
0.33
2.7*
0.43
2.6*
0.49
2.0
0.17
1.9*
0.25
3.1*
0.70
1.9*
0.23
1.9*
0.09
1.9
0.13
1.7*
0.14

1.5
0.29
1.8
0.19
1.7
0.13
2.4*
0.61
1.9
0.31
2.1*
0.52
2.8*
0.37
3.3
0.22
3.2*
0.34
2.0
0.49
2.7*
0.23
3.3*
0.18
3.5
0.32
3.4*
0.35

3.1
0.74
2.4
0.27
2.6
0.22
1.5*
0.63
2.2
0.47
1.4*
0.49
1.0*
0.33
0.6*
0.06
0.6*
0.08
1.7*
0.70
0.7*
0.14
0.6*
0.03
0.5
0.07
0.5*
0.10

67
17.9
74
7.3
64
9.5
252*
144.0
90
15.1
198
127.8
385
96.2
645
148.6
684*
106.9
223
153.6
545*
167.2
621*
65.5
689
127.2
891
591.2

33
11.5
59
15.4
40
12.9
165*
128.9
38
14.9
98*
72.5
275*
148.7
481
173.2
664*
327.7
88
81.0
537*
287.9
627*
187.7
624
115.2
1,281
1,665.2

13
1.0
13
1.4
13
2.8
13
3.7
14
2.2
11
1.8
16
2.8
23
3.5
31*
13.5
14
3.4
22*
9.1
50*
10.3
37
6.4
59*
23.3

0.3
0.05
0.3
0.04
0.2
0.05
0.3
0.04
0.3
0.04
0.3
0.05
0.3
0.06
0.6*
0.12
0.5*
0.6
0.3
0.08
0.6*
0.28
0.7
0.05
0.7*
0.15
0.6
0.07

128
26.2
87
17.5
120
10.8
72
21.2
115
20.2
43*
14.2
54*
10.8
35
11.9
30*
12.3
80
15.5
24*
6.2
24*
8.5
23
5.1
90
60.8

Note. M mean; SD standard deviation.

a
Results are expressed as mean values + SD (located below mean value).
Recovery phase. Number of animals per group 6, except n 5 for rats treated with 3 mg/kg/wk for 4 weeks with a 2 week recovery, and n 2 for rats treated with 3 mg/kg/wk for 6
weeks and no recovery.
*Significant at p  .05.
b

were no dexrazoxane-related alterations in the hematology data

from rats given dexrazoxane alone at 50 mg/kg/wk for 2 or 6
wks. Findings attributed to etoposide administration were
mainly limited to increased reticulocyte counts.
Clinical Chemistry
Progressive alterations in the clinical chemistry data in
doxorubicin-treated animals reflected both dose and duration
(Table 5). These changes included decreased serum total protein and albumin concentrations accompanied by increased
globulin concentrations (and corresponding decreased albumin
globulin ratios) when compared against concurrent vehicle
controls. These changes were first evident after 2 wks dosing
in animals given either 2 or 3 mg/kg/wk of doxorubicin and
at 6 wks in the animals at 1 mg/kg/wk. Serum triglyceride and
cholesterol concentration increases and alkaline phosphatase
activity decreases were first evident at 2 wks in animals given
3 mg/kg, 4 wks at 2 mg/kg, and 6 wks at 1 mg/kg. Increases in
serum urea and creatinine concentrations at 3 or 2 mg/kg/wk
after 2 or 6 wks of dosing, respectively, were also noted.
Generally, no recovery of the affected clinical chemistry parameters was observed during the respective off dose periods.

In general, the alterations in rats given doxorubicin (2 mg/

kg) in combination with dexrazoxane (50 mg/kg) weekly were
similar but of a lower magnitude of change after 6 wks
(Table 6). There were no alterations in the clinical chemistry
data from rats given dexrazoxane at 50 mg/kg/wk for 2 or 6
wks. There were no changes in the clinical chemistry data that
were attributed to the administration of etoposide, and no
treatment-associated changes in serum creatine kinase (total or
isoenzymes) in any group.
Assessment of cTnT (Roche) and cTnI (Beckman Access)
Concentrations
cTnI and cTnT were assessed in Groups 1 to 4 and 9 to 20. Predose data from all study animals were generally very consistent,
with cTnT concentrations at 0.01 ng/ml and cTnI concentrations
at 0.03 ng/ml. Two animals (1 each in doxorubicin Groups 3 and
4) showed relative elevation of cTn concentrations at predose
which remained increased at termination (wk 2; histologic minimal vacuolation or no histologic change were seen in the animals,
respectively). Minimal to mild elevations of cTn values in some
vehicle control animals at termination were apparent. Overall,
there was a good correlation (R 0.66) between the

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1154

REAGAN ET AL.

TOXICOLOGIC PATHOLOGY

TABLE 6.Changes in selected clinical chemistry parameters (M and SD) following treatment with etoposide, dexrazoxane, and dexrazoxane in
combination with doxorubicin.a

Dose mg/kg/wk
Etoposide 1

Weeks
of dosing
2
6

Etoposide 3

2
6

Dexrazoxane 50

2
6

Dexrazoxane 50
doxorubicin 2

2
6

Total
Albumin
protein g/dl
g/dl
6.0
0.35
6.4
0.23
6.1
0.16
6.3
0.33
6.0
0.33
6.5
0.63
5.9
0.35
5.3*
0.34

Globulin
g/dl

Albumin:
globulin

Cholesterol
mg/dl

Triglyceride
mg/dl

Urea
mg/dl

Creatinine
mg/dl

Alkaline
phosphatase U/L

1.5
0.28
1.9
0.21
1.6
0.13
1.9
0.14
1.5
0.24
1.8
0.43
1.6
0.26
2.8*
0.46

3.1
0.68
2.4
0.32
2.8
0.23
2.4
0.26
3.2
0.56
2.8
0.74
2.8
0.46
1.0*
0.47

63
18.3
73
10.9
66
20.0
74
15.8
55
16.1
70
10.5
68
18.5
341
242.9

42
20.5
79
18.1
33
15.0
69
15.0
38
9.6
75
21.6
29
5.7
326
330.2

13
1.9
12
1.0
13
2.1
13
1.4
11
1.6
12
1.5
14
3.5
14
6.3

0.3
0.05
0.3
0.0
0.3
0.05
0.3
0.0
0.3
0.05
0.3
0.04
0.3
0.0
0.3
0.17

122
12.5
85
19.7
112
15.1
90
15
144
21.2
92
9.3
91*
13.1
52*
14.1

4.5
0.15
4.5
0.18
4.5
0.08
4.5
0.31
4.6
0.16
4.7
0.28
4.3
0.19
2.6*
0.64

Note. M mean; SD standard deviation.

a
Results are expressed as mean values + SD (located below mean value). Number of animals per group 6.
*Significant at p  .05.

concentrations of cTnT and cTnI (Figure 1). Fold increase from

individual animal baseline was generally (but not always)
higher in cTnI values in comparison with cTnT concentrations
in most animals; whereas, this pattern tended to be reversed in
animals at the higher doxorubicin doses (2 or 3 mg/kg) at the
6-wk interval (of continuous dosing or following a 2-wk recovery period). Increase in magnitude of the mean signal and especially incidence of elevated cTn concentration was generally
observed with increasing dose and duration of doxorubicin
(Table 2). Partial or no recovery of cTn values following withdrawal of doxorubicin treatment was observed in animals
given either 2 or 3 mg/kg/wk.
A positive association between increase in terminal cTn value
and histologic findings was observed more frequently as the incidence of detected histologic change increased (Figure 1 and
Table 2). However, neither cTnI nor cTnT concentration was
well associated in absolute value or magnitude from baseline
with the extent of a histologic change. Increases in both cTnI and
cTnT concentration together in an animal also did not show
greater association with histologic change than cTnT concentration alone. The association between cTnI or cTnT concentration
increase and specific histologic location (atrial vs. myocardial)
could not be determined based on these data.
There was insufficient incidence of background histological
myocardial lesions to determine whether an association
between these background changes and increases in cTn concentration consistently occurred.
Assessment of High-sensitivity cTnI Concentrations
(Nanosphere)
A limited number of serum samples, collected at day 2 and
wk 4 (Groups 911, 15, 1724) and wk 2 and 6 (Groups 21

24), were available for assessment by the high-sensitivity cTnI

assay (Table 2). The serum cTnI concentrations were obtained
from samples that were approximately 2 years old and assay
precision in duplicates was variable (mean coefficient of variation 46%). At day 2, the majority of the study animals
showed cTnI concentrations <4 pg/ml. One animal given dexrazoxane alone at 50 mg/kg/wk for 6 wks demonstrated an
increased cTnI concentration throughout the course of the
study. Histologically, this animal showed a background change
of multifocal myocardial necrosis. Serum cTnI concentrations
in vehicle control males (Groups 9 and 17) ranged from 0 to
3.9 pg/ml, with the majority of values <3.0 pg/ml. Vehicle control animal data showed little variation between day 2 and wk
4. Among tested samples for animals administered doxorubicin, increase in incidence and mean serum cTnI concentration
(>4.0 pg/ml) was observed at wk 4, proportionate to the dose of
doxorubicin administered (Table 2). A relationship between
increased cTnI values at wk 4 and cardiac histopathology
(at wk 6) was generally observed in individual animals,
reflecting doxorubicin-associated vacuolation in the atrium
or ventricles or incidental findings of myocardial injury.
Additionally, in some animals (13/6) given a combination
of dexrazoxane and doxorubicin, elevations of cTnI concentrations at wks 2, 4, and 6 were noted and generally associated with either atrial or ventricular vacuolation. Figure 2
demonstrates these changes in rats given a weekly combination of dexrazoxane (50 mg/kg) and doxorubicin (2 mg/kg).
Samples from some (23/6) animals given dexrazoxane or
etoposide alone, serum cTnI concentrations with this assay
showed (mostly very small) increases >4.0 pg/ml at wk 2,
4, and/or 6 that were generally associated with background
histologic changes (focal to multifocal necrosis,
vacuolation).

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FIGURE 1.Comparison of cTnI and cTnT concentrations in control (A) and doxorubicin-treated (1 (B), 2 (C), or 3 (D) mg/kg/week for 2, 4, or 6
weeks [wks]) animals without recovery. Each animal is represented by an open symbol and if there are histological changes at 2, 4, or 6 wks they
are marked with a color (atrial/ventricular vacuolation [Vac.]) and/or a plus sign (necrosis). cTnI cardiac troponin I; cTnT cardiac troponin T.

Organ:Body Weights
Changes in organ weights not attributed primarily to
decreases in body weight were observed in animals given doxorubicin compared with concurrent vehicle controls (data not
shown). In these animals, relative (to body weight) spleen
weights were decreased at 2 or 3 mg/kg after 2 wks of dosing,
and 1 mg/kg after 6 wks of dosing (Groups 3, 4, and 18); liver
and kidney weights were increased at 2 or 3 mg/kg after 4 and
6 wks of dosing (Groups 13, 14, 19, and 20), organ:body
weight increases were also present after 4 wks recovery at 2
or 3 mg/kg (Groups 10 and 11) for liver and kidney, and at
3 mg/kg for other weighed organs (heart, spleen, and brain).

Statistical analysis of organ weights was not conducted for

animals given a 2-wk recovery after 4 wks of doxorubicin
dosing (Groups 15 and 16) due to the lack of concurrent vehicle controls.
Cardiac Histopathology
Vacuolation of atrial and ventricular cardiomyocytes was
present most consistently in animals treated with either doxorubicin or etoposide (Figure 3) and was generally absent from
vehicle control animals with the exception of 2 animals on
study for 6 wks with minimal atrial vacuolation where that
vacuolation was consistent with lipid. Vacuolation was most

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REAGAN ET AL.

FIGURE 2.Serial measurements of cTnI using the Nanosphere highsensitivity assay and cardiac histopathology results in rats given a
weekly combination of dexrazoxane (50 mg/kg) and doxorubicin (2
mg/kg).
Note. 3/6 animals had cTnI elevations associated with atrial or ventricular vacuolation typical of doxorubicin-induced injury and 1/6 animals had an elevation in cTnI associated with focal myocellular
necrosis. cTnI cardiac troponin I; WNL within normal limits.

TOXICOLOGIC PATHOLOGY

FIGURE 4.Focal cardiomyocyte necrosis with cellular fragmentation

typical of background lesions occasionally seen in control and treated
animals. Toluidine blue stain; original magnification 40.

Randomly present within doxorubicin and vehicle-treated

groups were individual animals with lesions not typical of
doxorubicin-induced cardiomyopathy. These lesions were generally minimal to mild and consistent with spontaneous background changes in laboratory rodents. More specifically,
these lesions were variably characterized as focal to multifocal
cardiomyocyte necrosis with or without mixed mononuclear
cell inflammation (Figure 4). A minimal focus of fibrosis was
present in the heart of one rat given 2 weekly doses of doxorubicin at 3 mg/kg and necropsied 1 wk after the second dose.
DISCUSSION

FIGURE 3.Cardiomyocyte vacuolation typical of doxorubicin cardiomyopathy. Discrete clear vacuoles distort individually affected cells.
Toluidine blue stain; original magnification 60.

prominent in animals given doxorubicin alone, but was also

present in some animals given etoposide alone and was mitigated in animals given doxorubicin and dexrazoxane. In the
former, minimal ventricular vacuolation was first seen in rats
given 2 weekly doses of 2 mg/kg doxorubicin and necropsied
1 wk after the second dose. Minimal atrial vacuolation was
present also at this time point in animals given 3 mg/kg. The
incidence and severity of both ventricular and atrial cardiomyocyte vacuolation increased with increasing cumulative
dose and duration of treatment.

In this rat model of chronic doxorubicin-induced cardiotoxicity, circulating cTnI and cTnT concentrations identified
cardiac injury at all doses (1, 2, or 3 mg/kg/wk). Both the
incidence and the mean magnitude of cTn signals generally
increased with increasing dose and/or longer duration of treatment. There was correlation between cTnT and cTnI signals in
evaluated samples. Serum cTn results following a 2- or 4-wk
recovery interval also indicated progression of cardiac injury
beyond the dosing period consistent with the recognized pathogenesis of doxorubicin cardiomyopathy. Overall, identification
of cardiac injury based on cTn increases was similar with both
assays. This is in contrast to the findings of Bertinchant et al.
who suggested that cTnT was a better indicator of cardiac
injury in rats treated with doxorubicin (Bertinchant et al.
2003); our investigation, as well as those of Bertinchant, used
a Beckman Access1 and Elecsys STAT1 Immunoassay to
quantitate cTnI and cTnT concentrations, respectively. In the
current study, the association between positive serum cTn
responses with these assays (values above the assay LOD) and
histological changes typical of doxorubicin-induced injury,
including vacuolation of the atria and ventricles (Herman et al.
1999), was greater at the higher doses and longer dosing

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CARDIAC TROPONIN AND DOXORUBICIN CARDIOTOXICITY

durations. Notably, the cTn concentration increased prior to the

onset of histologic lesions, but the magnitude of cTn absolute
value or change from baseline did not always match directly
with the grade of histologic change. The association between the
troponin concentration and histology may have been greater if
multiple sections of the heart were examined instead of a single
section.
Results of limited testing with the high-sensitivity Nanosphere assay also showed reproducible cTnI concentration
increases in wk 4 in animals treated with 2 or 3 mg/kg of doxorubicin. These responses were dose related in incidence and
mean magnitude and generally were associated with
doxorubicin-related histologic change subsequently identified
at termination. Increases (that were mostly small in magnitude)
were additionally seen in some animals dosed with dexrazoxane alone, or etoposide with apparent association to common
background histologic myocardial changes.
Due to sample volume constraints, the results of the highsensitivity cTnI assay and that of the other troponin assays
could not be directly compared for the same sampling time
points, but overall there was a general association of the
high-sensitivity assay results to the other cTn results obtained
for doxorubicin-administered animals. While our assessment
of the research assay was limited and the samples were over
2 years old, the precision of the assay was notably not as high
as has been reported for rat serum with another high-sensitivity
cTnI assay (Schultze et al. 2008). Overall, it has been shown
that cTnT is stable frozen for a least a year with a minimal
decrease (7%) after 2 years of storage (Basit et al. 2007). A
similar stability would be anticipated for cTnI and thus sample
stability likely did not affect the results.
This doxorubicin model of chronic, progressive low-level
cardiotoxicity can be contrasted with previously published
models of acute cardiotoxicity using isoproterenol (Clements
et al. 2010; York et al. 2007). The pathophysiology of cardiac
injury varies with these two cardiotoxicants and the histologic
lesions and serum cTn responses are distinct. Histologically,
chronic cardiotoxicity with doxorubicin administration results
in progressive vacuolar cardiomyocyte injury, whereas acute
cardiotoxicity of isoproterenol administration causes multifocal cardiomyocyte degeneration and necrosis in rats (and other
species). Accordingly, serum cTn in rats administered doxorubicin in this study showed progressive increases in cTn concentrations, all of relatively low magnitude; whereas a single dose
of isoproterenol administered to rats resulted in a rapid increase
in cTn concentration with return to baseline at 24- to 48-hr post
dose (Clements et al. 2010; York et al. 2007). These models of
cardiotoxicity with very different cTn response curves
illustrate the importance of understanding the expected pathophysiology of cardiotoxic injury when interpreting cTn results.
Doxorubicin toxicity also resulted in progressive and doserelated decreases in body weight, and changes in routine clinical pathology parameters; the latter included decreases in
white blood cell counts, RBC mass parameters, and serum
albumin, and increases in serum lipids as early as 2 wks at 2
or 3 mg/kg/wk. Indications of renal function deterioration

1157

(increases in serum urea and creatinine concentrations) were

observed at 2 or 3 mg/kg/wk after 6 or 2 wks of dosing, respectively. These collective changes reflected the well-established
characteristics of doxorubicin-induced gastrointestinal toxicity, hematoxicity, and nephrotoxicity (Bizzi et al. 1983; Tian
Hu, Brandle, and Zbinden 1983). Whether these toxicities had
impact on the myocardium and serum cTn concentrations,
especially at greater doses and dosing durations, cannot be
excluded. Throughout this study, the general protective effects
of dexrazoxane on doxorubicin-induced toxicity were apparent
based on lower magnitude of effect in most clinical pathology
parameters. Etoposide alone was associated with minimal
changes in clinical pathology parameters.
Results of this investigation support the utility of either
cTnT or cTnI testing to detect the chemically induced chronic
low-level cardiotoxicity. The incidence and mean magnitude of
cTnT or cTnI generally increased with increasing myocardial
injury. Notably, based on this model, association between cTn
increases and histologic findings can be less consistent with
very low-grade or early cardiotoxicity. As expected, serum cTn
increases were detected at earlier intervals than observed
histologic myocardial lesions. However, the magnitude of cTn
absolute value and change from baseline in individual animals
also did not match directly with the type or grade of histologic
change (in contrast to the models of acute myocardial toxicity).
The use of the newer cTnI high-sensitivity assay showed some
promise in detection of cardiac injury at even earlier intervals
and prior to histologic findings, than that of clinically approved
assays.
ACKNOWLEDGMENTS
The authors thank Rosemary Nicklaus for performing the
cTnI analysis on the Beckman Access1 Immunoassay System,
the colleagues at Covance Laboratories for conducting the
study, Dr. Steven Lipshultz and his laboratory at the University
of Miami for cTnT sample analyses, and Jennifer Pierson from
HESI for help in article preparation.
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